Isolation And Identification Of Bacterial Flora From Garri Sold At Ogige Market, Nsukka, Enugu State

Materials and methods

Materials used

Garri sample
Nutrient agar
Distilled water
Test tubes
Petri-dish
Glass spreader
Bunsen burner 
Cotton Wool

Collection of Garri samples

        With the purpose of isolating bacteria. Garri samples were collected from 20 different garri sellers in Nsukka within Nsukka market (ogige market). The samples were 10 samples of Red garri and 10 samples of white garri.

Media Reagent used

A Nutrient media is a general purpose medium supporting growth of a wide range of non-fastidious bacteria. It is useful because it remains solid even at relatively high temperatures.

The component of the media include:

5g peptone

3g beef extract/yeast extract

15g agar

Distilled water: 1 liter

NaCl: 5g

Final PH 6.8 at 25oc

Preparation of the media

A certain volume is of distilled water was poured in a conical flasks and a certain gram of the nutrient agar was weighed and dispenser into the conical flask containing distilled water. The mixture was shaken and then autoclaved at 121c for 15minutes. After autoclaving then allowed to cool a bit before dispensing into the petri-dishes.

Isolation of the organism

      After solidification of the agar and tested for sterility by incubating for 24hrs at room temperature before use.

From the serially diluted sample is being poured and spread on the plate and incubated for 24hrs in the incubator.

The plate were examined, and the verities of colonies present were noted. Discrete bacterial colonies which developed on the plates were then randomly, picked and purified by sub-culture into a free plate using the streak plate fetching. Discrete colonies which appeared on the plate were then transferred into the bijou bottle and stored as stock cultures for further tests.

Identification of isolates

            The isolates were examined microscopically for the colony morphology.

The identification procedures used include

Gram staining:

The procedure is rapid, simple and economical and provides useful information on the shape arrangement and biochemistry of the bacteria. The gram stain is used to divide bacteria into two broad groups, the Gram positive and the Gram negative bacteria.

Inoculation of stock culture

Nutrient media was prepared and poured into bijou bottle and autoclave at 121c for 15 minutes. Before cooling, the bottles were slanted and allow cooling. Then incubated for 24hours to check for sterility. The slants were inoculated using a wire loop, to make a smear with the organism on the media. Each isolate was inoculated on 4 bijou bottles and incubated in the incubator for 48 hours and store in the refrigerator.

Characterization of the isolates

Macroscopy:

Macroscopy was done by observing the morphology and cultural characteristics of the isolate on the nutrient media plate.

Microscopy:

Gram staining: This staining was done to know the organisms which retained or decolourised acetone alcohol based on the reaction, organisms were classified gram positive and gram negative on a clean glass slide, a smear of the organism was made in a drop of normal saline. The slide was allowed to air dry. The smear was then covered with crystal violet and allowed to stand for 30 seconds.
The slide was rinsed with distilled water and later covered with Lugo's iodine and allowed to stand for 60 seconds. The iodine was washed with water and decolourised with acetone alcohol and washed off with water immediately. It was then covered with safranine for 20 seconds and washed off with water and then allowed to dry. The slide is then observed with a microscope using X100 objective with oil immersion. A positive result will retain the acetone alcohol.

Indole Test

Materials: Kovac's reagent and peptone water

Certain species of bacteria are able to spilt the aromatic amino acid, tryptophan into indole, pyruvic acid and ammonia. The enzyme responsible for this reaction is known as Tryptophanse.

Tryptophan---------------indole_ pyruvic acid + Ammonia.                                                                                                                                                       

                        Tryptophanase                                               

A peptone broth is prepared by dissolving a certain gram of peptone powder into a distilled water.

Bijou bottles containing the stock samples is removed from the refrigerator. A wire loop is used to inoculated the organism into peptone water broth. The broth media is incubated at 37c for 48-96hrs. A 0.5 ml of Kovac's reagent is added and shakes gently. Pink/Red colour in the alcohol layer (top of the culture) indicates positive reaction.

Catalase Test

        Catalase test is carried out to demonstrate the presence of enzyme catalase that breaks down hydrogen peroxide to water and oxygen,

         H2O2-----------------H2O+ 1/2 O2 with a sterile were loop, a smear of the organism was made on a glass slide. A drop hydrogen peroxide was placed on the smeared organism the presence of effervescence (gas bubbles) shows positive and no effervescence shows negative reaction.

Oxidase Test:

      The oxidase reagent which is colourless but when oxidizes it turns to purple. Only organisms capable of producing peroxidase and hydrogen peroxide as a result of the presence of C-type cytochrome produced by the organism. On a piece of filter paper which was placed in a petridish, 2-3 drops of the oxidase reagent, Kovac's reagent was used in making a smear with the capillary end of pasture's pipette.

Sugar fermentation:

       The sugar used for the test was glucoses and manitol. The phenol red peptone water (oxiod) a nutrient base for fermentation studies was prepared according to the manufacture directions and poured into sterile bijou bottle. A Durham tube to be filled with the reagent. Discrete colonies of the test organism from the purified cultures were taken with sterile wire loop and inoculated into the test reagent and incubated for 37c for 24 hours. Fermentation colour change from wine-red to faint yellow indicate positive fermentation. While raised Durham tubes with air space indicate gas production.

Spore staining:

 A thick smear of the culture was made on a clean grease free slide and emulsified using normal saline, heat fixed and dried. It was flooded with malachite green and steamed for 4-5 minutes. Then, the stain was washed off with distilled water. After which, the slide was counterstained with safranin for 30 seconds. The stain was then washed off with distilled water blotted dry.

MOTILITY

The method used for the test is the hanging-drop method. Plasticine was used  to form  a ring on top of a clean slide. Then, a drop of the isolate which has already been inoculated in peptone water was placed on a clean cover slip. The slide containing the ring-like plasticine was then placed on top of the cover slip in such a way that the drop of the isolate, on the cover slip was in the middle of the ring. The slide, with the cover slip attached to it was quickly turned upside down such that the drop of the isolate hanged without touching the slide as a result of the plasticine ring which prevented the slide and the cover slip from meeting. The slide as then viewed under a microscope. A movement across the field of view indicated that the organism was motile.

OXIDASE TEST:

A filter paper was soaked with the oxidase reagent  (1%  tetramethyl-phenylenediamine aqueous solution) and a small amount of the isolate was picked with a sterile toothpick and put on top of the soaked filter paper. A positive reaction was indicated by a colour change to deep purple within 15 seconds while a negative isolate did not change colour.

CITRATE TEST:

This test isused to examine the ability of an organism to untilize citrate present in Simmon’s medium as a source of carbon for growth. The test isolates were inoculated directly on different Simmon’s citrate agar slants. The medium contains bromothymol blue which serves as indicator and incubated at room temperature for 24hours. A positive result was indicated by appearance of growth with blue colour from green colour.